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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-1823
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 3 1625-1631
Copyright © 2005 by The Endocrine Society

Recombinant Methionyl Human Leptin Administration Activates Signal Transducer and Activator of Transcription 3 Signaling in Peripheral Blood Mononuclear Cells in Vivo and Regulates Soluble Tumor Necrosis Factor-{alpha} Receptor Levels in Humans with Relative Leptin Deficiency

Jean L. Chan1, Stergios J. Moschos1, John Bullen, Kathleen Heist, Xian Li, Young-Bum Kim, Barbara B. Kahn and Christos S. Mantzoros

Division of Endocrinology, Diabetes, and Metabolism (J.L.C., S.J.M., J.B., K.H., Y.-B.K., B.B.K., C.S.M.) and Division of Immunology (X.L.), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Christos S. Mantzoros, M.D., D.Sc., Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, ST 816, Boston, Massachusetts 02215. E-mail: cmantzor{at}bidmc.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Studies of congenital complete leptin deficiency in animals and humans support a role for leptin in regulating immune function. Whether acquired relative leptin deficiency affects immunological parameters in healthy humans remains unknown. We thus used experimental models of relative leptin deficiency and recombinant methionyl human leptin (r-metHuLeptin) administration in humans to investigate whether r-metHuLeptin would activate signaling pathways in peripheral blood mononuclear cells (PBMCs) and whether acquired relative leptin deficiency and/or increasing circulating leptin levels into the physiologic range would change PBMC subpopulations and cytokines important in the T-helper cell and systemic immune responses. We found that r-metHuLeptin administration to healthy humans activates signal transducer and activator of transcription-3 signaling in PBMCs in vivo. Neither short-term leptin deficiency, induced by 3-d complete fasting, nor physiologic r-metHuLeptin replacement for the same period of time had a major effect on PBMC subpopulations or serum cytokines in healthy men. In contrast, normalizing serum leptin levels over 8 wk in lean women with relative leptin deficiency for 5.1 ± 1.4 yr (mean ± SE) due to chronic energy deficit increased soluble TNF{alpha} receptor levels, indicating activation of the TNF{alpha} system. These findings suggest that relative leptin deficiency due to more long-term energy deprivation is associated with defects in immunological parameters that may be corrected with exogenous r-metHuLeptin administration. Further studies are warranted to assess the implications of acquired relative hypoleptinemia and/or r-metHuLeptin administration on the immunosuppression associated with energy- and leptin-deficient states in humans.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
LEPTIN, AN ADIPOCYTE-SECRETED hormone with structural similarity to cytokines, circulates in the serum in proportion to the amount of energy stored in adipose tissue and/or acute changes in energy availability (1, 2, 3). As a signal of energy deficiency, leptin has been shown to play an important role in regulating the neuroendocrine response to short-term caloric deprivation in rodents (4) and humans (5) by reversing starvation-induced changes in their neuroendocrine axes (4, 5). Recently we have also shown that recombinant methionyl human leptin (r-metHuLeptin), administered in physiologic replacement doses, significantly improves neuroendocrine and reproductive function in women with long-term energy deficit and relative leptin deficiency for several years (6).

Immune function is another energy-dependent process that is altered in states of energy deficit, such as malnutrition (7). In vitro studies as well as in vivo studies in rodents support a role for leptin in regulating immunological processes. Human peripheral blood mononuclear cells (PBMCs) express the long isoform of the leptin receptor (8, 9), which is also highly expressed in the hypothalamus. In human PBMCs in vitro, leptin activates the Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway for signal transduction and enhances proliferation and activation of monocytes and T lymphocytes (10, 11, 12). In addition, leptin-deficient ob/ob and db/db mice with mutations in the leptin and leptin receptor genes, respectively, have impaired cell-mediated immunity (13, 14, 15), and leptin administration to ob/ob mice substantially increases thymic cellularity (16). In normal mice, falling leptin levels mediate the starvation-induced immunosuppression, and exogenously administered leptin reverses the marked reductions in CD4+CD8+ thymocytes (16) and delayed-type hypersensitivity responses in this model (8).

Similar studies, albeit uncontrolled, in human models of congenital complete leptin deficiency also provide compelling preliminary evidence for the importance of leptin in immunological function in vivo. In an extended Turkish family that included members with morbid obesity resulting from a leptin gene mutation, a higher childhood mortality rate due to infections was noted in several obese members with presumed leptin deficiency, compared with normal-weight, presumably wild-type siblings (17). Moreover, leptin administration to two children with proven congenital leptin deficiency improved CD4+ T-helper cell counts, T-cell proliferation, and cytokine release (18). Although the complete congenital absence of leptin in humans can significantly perturb immunological processes, and observational data in malnourished infants and fasting patients with rheumatoid arthritis have linked low leptin levels with suppression of CD4+ T-cell activation and lymphoproliferation (19, 20), the effect of relative leptin deficiency and subsequent leptin replacement on immune function in humans has not yet been directly evaluated in the context of placebo-controlled leptin administration trials. Such information has important clinical relevance for conditions of energy deficiency, including malnutrition, that are associated with diminished immune responses as well as autoimmune diseases that may be influenced by leptin (19, 21, 22).

To address these questions, we performed interventional studies involving administration of r-metHuLeptin or placebo to humans with relative hypoleptinemia to test the following hypotheses. First, we sought to evaluate whether, similar to prior in vitro studies, leptin can activate signal transduction pathways in vivo in immune cells from healthy humans and whether this occurs in a dose-dependent manner. Then, to determine whether short and/or more long-term relative leptin deficiency affects immunological parameters in humans, we studied the effect of energy deprivation-induced leptin deficiency with placebo or physiologic r-metHuLeptin administration on several parameters of immunological function. These parameters include PBMC subpopulations, cytokines important in T-helper cell responses, and the TNF{alpha} system because TNF{alpha} represents an important link between specific immune responses and systemic inflammatory responses.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Human subjects

The study protocols were approved by the Institutional Review Board of the Beth Israel Deaconess Medical Center (BIDMC), and subjects gave written informed consent to participate. Clinical-quality r-metHuLeptin was supplied by Amgen, Inc. (Thousand Oaks, CA) and administered under an Investigational New Drug application submitted to the Food and Drug Administration (C.S.M.). All subjects were healthy, had no evidence of immunologic or endocrine disease based on physical examination and routine blood tests, and had no history of recent infection.

Study 1: Leptin signaling in PBMCs in vivo in response to physiologic and pharmacologic doses of r-metHuLeptin

Five lean men [age 22.2 ± 0.9 (mean ± SE) yr, body mass index (BMI) 22.0 ± 0.5 kg/m2] and five overweight men (age 23.4 ± 1.5 yr, BMI 32.0 ± 1.0 kg/m2) were studied in the fed state (on an isocaloric diet) two separate times (separated by 6.4 ± 1.0 wk) in the BIDMC General Clinical Research Center (GCRC). They received a single dose of r-metHuLeptin by sc injection at 0800 h: 0.01 mg/kg during the first study and 0.3 mg/kg during the second study. Blood samples to evaluate JAK-STAT, MAPK (a protein stimulated by leptin in other tissues and cell lines) (23), and B-cell leukemia-2 (Bcl-2) (a protein important in regulating apoptosis in ß-cells) expression and phosphorylation in PBMCs were obtained just before the 0.01 mg/kg dose (at 0800 h), 2 h after the 0.01 mg/kg dose (at 1000 h), and 2 h after the 0.3 mg/kg dose (at 1000 h).

Study 2: Effect of short-term leptin deficiency and administration of placebo vs. physiologic replacement r-metHuLeptin doses on PBMC subpopulations and serum cytokine levels in men with hypoleptinemia induced by fasting for 3 d

Eight lean men (age 23.3 ± 1.2 yr, BMI 23.7 ± 0.6 kg/m2) participated in a study in the BIDMC GCRC involving complete fasting for 3 d with administration of placebo (expected to result in leptin levels approximately 30% of baseline), low-dose r-metHuLeptin (to achieve leptin levels approximately 50% of baseline), or replacement-dose r-metHuLeptin (to fully correct leptin levels to physiologic fed-state levels). Each of the fasting studies was separated by at least 7 wk. One subject participated in only the study of fasting with placebo, one subject did not participate in the replacement-dose r-metHuLeptin study, and another subject did not return for the low-dose r-metHuLeptin study. During the fasting studies, subjects received only caffeine-free and calorie-free liquids and NaCl (500 mg), KCl (40 mEq), and a standard multivitamin with minerals daily. Blood samples were obtained at 0800 h on d 1 and 4 to measure PBMC subpopulations and on d 1 and 3 for measurement of leptin and serum cytokines: interferon (IFN)-{gamma}, IL-4, IL-10, TNF{alpha}, soluble TNF{alpha} receptor-I (sTNF-RI), and soluble TNF-{alpha} receptor-II (sTNF-RII). For sTNF-RI and sTNF-RII, serum was available for only five subjects who participated in all three studies. For the low-dose r-metHuLeptin admission, r-metHuLeptin was administered as four sc injections per day at doses ranging from 0.001 to 0.008 mg/kg·d, based on the subject’s baseline leptin level. For the replacement-dose r-metHuLeptin admission, r-metHuLeptin was administered at a dose of 0.04 mg/kg·d on the first day and 0.1 mg/kg·d on the second day, which was designed on the basis of prior pharmacokinetic studies to achieve physiologic serum leptin levels similar to that in the fed state (24). The total daily r-metHuLeptin leptin dose for each day was divided into four equal doses given every 6 h.

Study 3: Effect of physiologic replacement r-metHuLeptin administration on serum cytokine levels in women with long-term relative leptin deficiency

Eight lean women (age 24.8 ± 1.9 yr, BMI 20.5 ± 0.7 kg/m2) with chronic energy deficit, relative leptin deficiency (baseline leptin level 3.0 ± 0.7 ng/ml), and hypothalamic amenorrhea (5.1 ± 1.4 yr duration) related to strenuous exercise or low weight received r-metHuLeptin (0.08 mg/kg·d) sc twice daily for 8 wk, with 40% of the daily dose at 0800 h and 60% at 2000 h to mimic normal diurnal variation of leptin levels. Blood samples for measurement of leptin and serum cytokines: IFN{gamma}, IL-4, IL-10, sTNF-RI, and sTNF-RII were obtained just before initiation of r-metHuLeptin and after 2, 4, 6, and 8 wk of treatment (serum for IFN{gamma} and IL-4 were obtained at 1, 3, 5, and 7 wk of treatment). One subject participated in the study for only 4 wk for reasons unrelated to the study (moved overseas). For comparison, the same cytokines were measured in a control group of seven healthy eumenorrheic lean women of similar age and BMI (age 22.4 ± 1.1 yr, BMI 21.5 ± 0.8 kg/m2) who received no intervention. Percent fat mass was measured by bioelectric impedance analysis (BIA, RJL Systems, Quantum Township, MI) in both groups and also by dual-energy x-ray absorptiometry (Hologic, Waltham, MA) in the amenorrheic subjects.

Measurements

Western blots for the signaling study. Blood was collected in heparin-containing tubes, kept on ice, and processed within 1 h. PBMCs were isolated by separation over standard Ficoll-Paque (Pharmacia, Piscataway, NJ) density gradient centrifugation (2700 rpm, 40 min), and the interface was washed twice with RPMI 1640/10% fetal bovine serum (Life Technologies, Inc./BRL, Gaithersburg, MD) (1700 rpm, 5 min). Twenty-five micrograms of PBMC lysates protein per lane were resolved by SDS-PAGE (4–15% gel) and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). The nitrocellulose membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with phospho-specific STAT3 (Tyr705), STAT1 (Tyr701), and STAT5 (Tyr694) polyclonal antibody (Cell Signaling Technology, Beverly, MA), active MAPK polyclonal antibody (Promega, Madison, WI) with dually phosphorylated The/Glu region (pTEpY) derived from the active form of MAPK enzymes, or phospho-specific Bcl-2 (Ser70) polyclonal antibody (Cell Signaling Technology) in 1% nonfat dry milk overnight at 4 C. The membranes were washed, as previously described (23). The bands were visualized using the enhanced chemiluminescence system (Amersham, Arlington Heights. IL) and quantified by densitometry (Molecular Dynamics, Sunnyvale, CA).

PBMC subpopulations. Flow cytometric analysis of isolated PBMCs was carried out by a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The following fluoroscein isothiocyanate (FITC) and/or phycoerythrin (PE)-conjugated monoclonal mouse antihuman antibodies were used for lymphocyte subset determination: CD3-PE, CD8-PE (Coulter Corp., Miami, FL), CD4-PE, CD19-PE, CD45RA-FITC, CD45RO-FITC (BD PharMingen, San Diego, CA). PBMCs were gated and determined by both forward-light and side-scatter intensity. Analysis was performed on at least 10,000 cells with the CellQuest software (Becton Dickinson). Isotype-matched control antibodies were included in the staining as controls.

Assays

Leptin was measured using RIA [sensitivity 0.5 ng/ml and coefficient of variation (CV) 6–7%, Linco Research, St. Charles, MO]. Cytokines were measured in serum or plasma using commercially available ELISAs: TNF{alpha} (Quantikine HS, sensitivity 0.06 pg/ml, CV 5.3–8.8%, R&D Systems, Minneapolis, MN); sTNF-RI (Quantikine, sensitivity 3 pg/ml, CV 2.7–6.9%, R&D Systems); sTNF-RII (Quantikine, sensitivity < 1 pg/ml, CV 1.6–2.5%, R&D Systems); IFN{gamma} (high sensitivity 0.1 pg/ml, CV < 10%, Amersham Pharmacia Biotech, Piscataway, NJ); IL-4 (sensitivity 0.2 pg/ml, Research Diagnostics, Inc., Flanders, NJ); IL-10 (high sensitivity 0.1 pg/ml, CV < 10%, Amersham).

Statistical analysis

Data are presented as mean ± SE. For the signaling study (study 1), mean ratios of phosphorylated to total protein levels were compared among baseline, after 0.01 mg/kg r-metHuLeptin, and after 0.3 mg/kg r-metHuLeptin using one-way ANOVA with post hoc tests by least significant differences. For the fasting and leptin replacement study (study 2), nonparametric Wilcoxon signed ranks tests were used as primary analysis and paired t tests as secondary analysis to compare leptin and cytokine levels and PBMC subpopulations at baseline (d 1) and post intervention (d 3 or 4). To adjust for multiple comparisons in study 2, P < 0.017 (using Bonferroni correction) was considered significant and 0.017 ≤ P < 0.05 was considered borderline significant. For the leptin replacement study in women (study 3), nonparametric Wilcoxon rank sum tests were used as primary analysis and independent samples t tests as secondary analysis to compare leptin and cytokine levels and body composition parameters between amenorrheic subjects and controls. In amenorrheic subjects, a repeated-measures analysis was used to test for changes in cytokine levels across time (general linear model in SPSS version 8.0, SPSS, Chicago, IL) using either a linear or quadratic model according to the best fit of the data, with an overall P value reported for change across the entire study. Post hoc tests were used to compare average values from baseline to each time point during treatment. Similar results were obtained using nonparametric and parametric tests except where noted. Analyses were carried out using StatView 5 (SAS Institute Inc., Cary, NC) and/or SPSS (version 8.0, SPSS).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Leptin signals in PBMCs through phosphorylation of STAT3 but not STAT1, STAT5, MAPK, or Bcl-2

We first investigated whether leptin can signal in PBMCs through the STAT, MAPK, and/or Bcl-2 signaling pathways by measuring the phosphorylation of STAT1, STAT3, STAT5, MAPK, and Bcl-2 in PBMCs after administration of physiologic and pharmacologic doses of r-metHuLeptin in a group of healthy men. We compared data from lean and obese subjects to assess whether obesity, considered a leptin-resistant state, may affect the ability of leptin to signal due to possible receptor or postreceptor defects in obese subjects. At baseline, STAT3 phosphorylation was not different between lean and obese men [40.2 ± 6.2 arbitrary units (AU) vs. 46.0 ± 16.1 AU, P = 0.81]. In lean men, the phosphorylation of STAT3 increased from 40.2 ± 6.2 AU at baseline to 107.2 ± 11.9 AU after a low physiologic dose (0.01 mg/kg) of r-metHuLeptin and increased further to 186.7 ± 27.6 AU after a pharmacologic dose (0.3 mg/kg) (P = 0.004 by ANOVA; Fig. 1Go). STAT3 phosphorylation was significantly higher after the pharmacologic dose of r-metHuLeptin, compared with baseline (P = 0.001) and compared with the low physiologic dose (P = 0.03) by post hoc tests. STAT3 phosphorylation after the low physiologic dose tended to be higher, compared with baseline (P = 0.07).



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  		FIG. 1. STAT3 phosphorylation in PBMCs in lean (n = 5) and obese men (n = 5) at baseline before r-metHuLeptin administration (indicated by 0 mg/kg), 2 h after 0.01 mg/kg r-metHuLeptin administration, and 2 h after 0.3 mg/kg r-metHuLeptin (P = 0.004 for lean men, P = 0.028 for obese men, by ANOVA). *, P < 0.05 vs. baseline. ^, P < 0.05 vs. 0.01 mg/kg r-metHuLeptin dose. Proteins (25 µg) were separated by SDS-PAGE on 4–15% gels and transferred to nitrocellulose membrane. Phosphorylated STAT3 was visualized by immunoblotting with antibody specific for phospho-STAT3. Bands were quantitated using a densitometer. STAT3 phosphorylation was normalized by the level of total STAT3. Bars represent the means of three different measurements using the same samples.

 
For the obese men, STAT3 phosphorylation increased similarly from 46.0 ± 16.1 AU at baseline to 125.5 ± 35.9 AU after the low-dose and 178.1 ± 21.9 AU after 0.3 mg/kg r-metHuLeptin (P = 0.03 by ANOVA; Fig. 1Go). By post hoc tests, STAT3 phosphorylation was higher after the pharmacologic dose, compared with baseline (P = 0.009) but was not significantly different from the low physiologic dose (P = 0.26). There were no significant changes in STAT1, STAT5, MAPK, or Bcl-2 phosphorylation with r-metHuLeptin administration at the physiologic or pharmacologic doses (data not shown).

Effect of short-term leptin deficiency and administration of placebo vs. physiologic replacement r-metHuLeptin doses on PBMC subpopulations and serum cytokine levels in lean men

Because leptin signals in lymphocytes, we then evaluated whether decreasing leptin levels by fasting for 3 d to 30% of baseline and/or restoring leptin levels by administration of r-metHuLeptin at either low (50% of baseline) or full replacement doses during fasting would affect PBMC subpopulations and/or serum cytokine levels in healthy humans. We measured cytokines that are important in the CD4+ T-helper cell response, including proinflammatory cytokines produced by Th1 cells and regulatory/antiinflammatory cytokines secreted by Th2 cells. These include IFN{gamma}, a proinflammatory Th1 cytokine that determines Th1 differentiation of CD4+ naive T-cells; IL-4, a regulatory Th2 cytokine that causes Th2 differentiation and inhibits the generation of IFN{gamma}-secreting cells; and IL-10, another regulatory cytokine important in both the Th1 and Th2 response. We also evaluated the effect of leptin on TNF{alpha}, which is important in both T-helper and systemic inflammatory responses, and on sTNF-RI and sTNF-RII, which reflect activation of the TNF{alpha} system (25). In animals and humans, TNF{alpha} increases leptin levels (26, 27), and observational studies in humans have demonstrated a positive association between leptin and soluble TNF{alpha} receptors (25, 28, 29).

In lean men, fasting significantly decreased leptin levels to approximately 30% of baseline by the third day (Table 1Go). Low-dose r-metHuLeptin administered during fasting partially restored the fasting-induced decline in leptin levels to approximately 60% of baseline, whereas replacement-dose r-metHuLeptin fully restored leptin levels to levels that were slightly higher than those observed in the fed state but within the physiologic range for lean men (Table 1Go). Body weight decreased by approximately 2 kg during each fasting study with no differential effect of r-metHuLeptin.


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  		TABLE 1. Serum leptin levels, PBMC subpopulations, and serum cytokine levels important in the T-helper cell response and the TNF-{alpha} system in eight lean men at baseline (d 1) and after 2 d of fasting (d 3)1 with administration of placebo (P), low-dose r-metHuLeptin (LL), or replacement dose r-metHuLeptin (RL)

 
Complete fasting decreased the total T-lymphocyte (CD3+) count to a similar degree in all three fasting studies, although the decrease was not significant when replacement-dose r-metHuLeptin was administered during fasting (Table 1Go). The CD4+ T-helper cell count showed a similar borderline significant decrease in response to fasting, with no differential effect of r-metHuLeptin on naive (CD45RA+) and memory (CD45RO+) CD4+ T cells. The percentage of CD4+ T cells tended to decrease during fasting (P = 0.03) but not when low-dose (P = 0.34) or replacement-dose r-metHuLeptin (P = 0.21) was administered during fasting. However, the magnitude of the decrease in percent CD4+ T cells was again similar in all three fasting studies. Otherwise, PBMC subpopulations (including CD8+ T cells) were not affected by fasting and/or r-metHuLeptin replacement (data not shown for other subsets).

There were no significant changes in T-helper cell cytokines (IFN{gamma}, IL-4, IL-10) or the TNF{alpha} system, except for a borderline significant increase in TNF{alpha} (P = 0.02) during fasting with low-dose r-metHuLeptin and similar borderline decrease in sTNF-RI (P = 0.04) during fasting alone, which did not occur when r-metHuLeptin was administered during fasting (Table 1Go). However, the magnitude of the decrease in sTNF-R1 levels was similar to the decrease observed when r-metHuLeptin was administered during fasting. These data indicate that relative leptin deficiency for 3 d may have a minor effect on cytokine levels that is not of a magnitude sufficient to achieve statistical significance after adjustment for multiple comparisons. Additionally, there was no significant differential effect of r-metHuLeptin on the above parameters during this short period of time.

Effect of physiologic replacement r-metHuLeptin administration on serum cytokine levels in women with long-term relative leptin deficiency

Because short-term leptin deficiency (due to complete fasting) over the course of a few days did not have major effects on PBMC subpopulations and markers of the T-helper response, we then investigated whether a longer duration of leptin deficiency and/or leptin replacement may be required for effects on immune function to be evident. We studied women with a several-year history of energy deficit (excessive energy output vs. input) and amenorrhea related to low leptin levels, i.e. a model of more chronic, relative leptin deficiency, and administered physiologic-dose r-metHuLeptin replacement for 8 wk to these women. Compared with normal control women without leptin deficiency, subjects were similarly lean (BMI of amenorrheic subjects: 20.5 ± 0.7 kg/m2 vs. controls: 21.5 ± 0.7 kg/m2, P = 0.30) but had significantly lower leptin levels (amenorrheic subjects: 3.0 ± 0.7 ng/ml vs. controls: 11.4 ± 1.6 ng/ml, P = 0.003) and percent fat mass [amenorrheic subjects: 23.7 ± 1.4% (BIA), 22.4 ± 1.3% (dual-energy x-ray absorptiometry) vs. controls: 29.9 ± 1.4% (BIA), P = 0.02 vs. controls by BIA].

In amenorrheic subjects, circulating leptin levels increased significantly over 8 wk of r-metHuLeptin treatment (P < 0.007), and, starting at 4 wk and continuing until 8 wk of treatment, leptin levels were not significantly different from that of controls (Table 2Go). At baseline, serum IL-10 levels in amenorrheic subjects were similar to controls and did not change with r-metHuLeptin treatment (Table 2Go). Serum TNF{alpha}, IFN{gamma}, and/or IL-4 levels were generally below the limit of detection for amenorrheic subjects and/or controls (data not shown). At baseline, sTNF-RI and sTNF-RII were significantly lower in amenorrheic subjects, compared with controls (Table 2Go). During treatment with r-metHuLeptin, sTNF-RI levels increased significantly (P < 0.001) with a peak at 6 wk of treatment to levels that were not different from controls (Table 2Go). sTNF-RII levels also increased with r-metHuLeptin (P = 0.02) with a peak at 4 wk, although levels remained lower than that of controls over the course of the study (Table 2Go).


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  		TABLE 2. Serum leptin levels and cytokines in seven normal control women and in eight amenorrheic women with relative leptin deficiency at baseline and after 2, 4, 6, and 8 wk of r-metHuLeptin replacement (0.08 mg/kg)

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Nutritional status is important for the integrity of the immune system, as evidenced by the increased rate of infections and delayed wound healing in low-leptin states of energy deficiency, including starvation (30). The capacity to mount an immune response is an energy-dependent process and thus diminished in the setting of malnutrition. It has been proposed on the basis of animal studies (8) that low levels of leptin, an adipocyte-secreted hormone that signals energy deficit, may play a role in the immunosuppression associated with starvation (31). Uncontrolled studies of rare cases of humans with lifelong congenital leptin deficiency support this notion (17, 18), but whether the same hypothesis holds true for relative leptin-deficient states in humans, an area of substantially broader scope and significant clinical relevance, remains unknown. To address this question, we performed interventional studies in humans to assess whether r-metHuLeptin administration in vivo activates signaling pathways in PBMCs and whether increasing leptin levels through exogenous r-metHuLeptin improves markers of immune function in states of short-term as well as relatively more long-term energy deficit, including PBMC subpopulations and cytokines important in the T-helper cell response.

We first investigated whether leptin signals in vivo in cells important in the immune system. Previous studies have shown that the long isoform of the leptin receptor (ObRb) that is primarily involved in signal transduction through activation of the JAK-STAT system is expressed not only in the hypothalamus in which leptin exerts its main effects on energy homeostasis but also in PBMCs (9, 12). In vitro, leptin enhances proliferation of human PBMCs in a dose-dependent manner and induces monocytes to produce the proinflammatory cytokines TNF{alpha} and IL-6 (11, 12). The proliferative effect of leptin on lymphocytes in mice is mediated by a specific effect on leptin receptor signaling, rather than through a nonspecific mitogenic stimulus (8). Furthermore, leptin in vitro has been shown to activate the JAK-STAT signaling pathway in human PBMCs through the tyrosine phosphorylation of JAK-2/3 and STAT3 (10) and to have a trophic effect on monocyte survival through the MAPK pathway (32). In this study, we show for the first time that r-metHuLeptin in vivo in humans increases STAT3 but not STAT1, STAT5, MAPK, or Bcl-2 phosphorylation in PBMCs. Further studies are warranted to evaluate whether other signaling pathways are similarly activated in PBMCs and whether alterations in intracellular signaling correlate with changes in immunological parameters, e.g. cytokine production by T-helper cells.

Previous mechanistic studies have also shown that leptin modulates T-helper cell function and phenotype. In vitro, leptin influences human CD4+ T cells toward a proinflammatory Th1 phenotype by stimulating production of cytokines such as IL-2 and IFN{gamma} (12). Similarly, leptin administration in vivo to mice during 48 h of starvation (8) and to fed nonobese diabetic mice (33) biases the T-helper cell response toward a Th1 phenotype and away from Th2 cells that secrete cytokines with predominantly regulatory function (such as IL-4 and IL-10). The distinction between Th1 and Th2 is not necessarily always completely distinct in humans in vivo, however, indicating the complexity of the immune response. Humans with congenital leptin deficiency have decreased production of cytokines important in both Th1 and Th2 functions, including IFN{gamma}, IL-4, and IL-10, and a corresponding marked increase in all of these cytokines after r-metHuLeptin treatment (18). The importance of leptin in T-helper cell responses has significant clinical relevance because the T-helper response is critical to many aspects of the immune response including cellular and antibody-mediated immunity and the ability to respond to intracellular microbes (bacteria, protozoa, and fungi) and viruses (34, 35).

Suppression of the immune response is common in states of malnutrition (36). In conjunction with this immunosuppression, malnourished infants have been shown to have low leptin levels, and a 10% weight gain resulting in a significant increase in leptin levels also resulted in a significant enhancement of the immune response (19). Conversely, leptin has been shown to play a pathogenic role in experimental animal models of autoimmunity, including encephalomyelitis, intestinal inflammation, antigen-induced arthritis, and type 1 diabetes (22, 33, 37, 38, 39, 40). Although the role of leptin in the pathogenesis of autoimmune diseases in humans remains to be more fully elucidated, prolonged fasting for 7 d in rheumatoid arthritis patients decreased markers of CD4+ lymphocyte activation and increased IL-4 production (20). We thus studied directly whether acute short-term starvation, i.e. complete fasting for 3 d, resulting in short-term leptin deficiency, would affect markers of the T-cell response in healthy lean men. Using this paradigm, we observed a modest decrease of the T-lymphocyte subpopulation with fasting, but restoring leptin to physiologic, fed-state levels with exogenous r-metHuLeptin administration for 3 d did not have a substantial effect on either PBMC subpopulations or circulating levels of T-helper cytokines. It remains possible that more detailed and sensitive measurements of immune function, such as lymphocyte proliferation and cytokine production by PBMCs, are required to detect subtle changes in immune function in the setting of relative leptin deficiency of short duration. However, these findings are in distinct contrast to the marked effect of lifelong, complete congenital leptin deficiency on immune function, as manifested by a presumed higher infection-related mortality rate (17) and diminished CD4+ T-cell counts and cytokine production, and the dramatic effect of subsequent leptin replacement on these parameters (18). Thus, exposure of the immune system to more long-term and pronounced energy (and consequently leptin) deficiency may be required for changes in immune function to be evident.

In this regard, it was useful to employ an experimental paradigm of leptin deficiency of intermediate duration between acute fasting in healthy humans and complete chronic leptin deficiency, i.e. strenuously exercising women athletes with relative leptin deficiency due to chronic energy deficit (excessive energy output vs. input) (41, 42). These women, who also had hypothalamic amenorrhea for a mean of 5.4 yr, were otherwise healthy with no clinically apparent deficiencies in immune function, although more detailed evaluation of immune status is required to elucidate this further. States of more severe energy deficit, e.g. eating disorders such as anorexia nervosa, are associated with alterations in cell-mediated immunity and cytokine production (43, 44). We thus evaluated the TNF{alpha} system, a more easily evaluable systemic marker of immune function that has also been shown to have a positive association with leptin in observational studies (25, 28, 29). TNF{alpha} is the principal mediator of the host response to Gram-negative bacteria and may also play a role in responses to other infectious organisms. TNF{alpha} mediates both natural and acquired immunity and is an important link between specific immune responses and acute inflammation. Serum TNF{alpha} levels are often difficult to measure in healthy individuals, and thus soluble TNF{alpha} receptor levels, which act as a buffer to prolong the biological effects of TNF{alpha}, reflect more accurately the degree of activation of the TNF{alpha} system (25, 45). We found that soluble TNF{alpha} receptor levels were significantly lower at baseline in women with chronic relative leptin deficiency, compared with control women with higher leptin levels. Low activity of the TNF{alpha} system could potentially be associated with defects in immune responses to infection, although this requires further study in the future. With physiologic-dose r-metHuLeptin replacement over a few months, levels of the soluble TNF{alpha} receptors increased to ranges lower than or similar to those observed in controls but did not reach the high levels observed in obese women with diabetes and increased risk of cardiovascular disease (29, 46).

In summary, we found that r-metHuLeptin signals through the JAK-STAT signaling system in human PBMCs in vivo, suggesting that leptin’s effects on immune function are at least in part direct through activation of specific leptin receptors on PBMCs. Neither fasting-induced hypoleptinemia nor r-metHuLeptin administration at physiologic doses for 3 d has a major effect on PBMC subpopulations or serum cytokine levels over the short term, although possible local paracrine effects require further evaluation. These data stand in distinct contrast to the beneficial effect of leptin replacement on immune function parameters in children with long-standing complete leptin deficiency and the demonstration herein that r-metHuLeptin administration in replacement doses over a few months increases activity of the TNF{alpha} system in women with hypothalamic amenorrhea and long-term relative leptin deficiency. Thus, it appears that duration of hypoleptinemia is of importance in the development of immunological abnormalities. Whether a threshold effect for leptin exists, i.e. whether a critically low leptin level needs to be present, in addition to a certain duration of time, for abnormalities in immune function to be evident, and what these specific thresholds are in terms of time course and dose response require further study. These results may have potentially important implications for other relative leptin-deficient states, e.g. anorexia nervosa, chronic malnutrition, or the cachexia of chronic diseases such as HIV/AIDS and cancer. These data may also have relevance for autoimmune diseases associated with hyperleptinemia, and further study is warranted to evaluate whether decreasing leptin levels in these conditions may prove to be beneficial.


   Acknowledgments
 
We are indebted to Alex M. DePaoli, M.D., and Amgen, Inc. for providing r-metHuLeptin for these studies; the General Clinical Research Center (GCRC) nurses at Beth Israel Deaconess Medical Center for collecting the samples for this research; and the GCRC nutritionists for their help with the isocaloric and fasting diets.


   Footnotes
 
This work was supported by National Institutes of Health (NIH) Grants MO1-RR01032 and R01-58785, NIH Grants RO1-DK43051 and DK56116 (to B.B.K.), and K23 RR018860 (to J.L.C.), a grant from Amgen Inc. (to C.S.M.), and a grant from the American Diabetes Association (7-02-JF-26 to Y.-B.K.).

First Published Online December 21, 2004

1 J.L.C. and S.J.M. contributed equally to the work presented herein. Back

Abbreviations: AU, Arbitrary unit; Bcl-2, B-cell leukemia-2; BIA, bioelectric impedance analysis; BMI, body mass index; CV, coefficient of variation; FITC, fluoroscein isothiocyanate; IFN, interferon; JAK, Janus kinase; PBMC, peripheral blood mononuclear cell; PE, phycoerythrin; r-metHuLeptin, recombinant methionyl human leptin; STAT, signal transducer and activator of transcription; sTNF-RI, soluble TNF{alpha} receptor-I; sTNF-RII, soluble TNF-{alpha} receptor-II.

Received September 14, 2004.

Accepted December 14, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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